Difference between revisions of "Part:BBa K1199044"
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[[File:T--UESTC-China--W249P-P84A-W.png|700px|thumb|center|'''Figure 1'''.The activity of mutants on 2,3-DCP Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃]]
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[[File:https://static.igem.org/mediawiki/2017/2/2b/T--UESTC-China--W249P-P84A-W.png|700px|thumb|center|'''Figure 1'''.The activity of mutants on 2,3-DCP Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃]]
  
  

Revision as of 08:20, 30 October 2017

HheCW249P 2,3-DCP(2,3-dichloropropanol)->Glycerol

HheC displayed high regioselectivity and moderate to high enantioselectivity that can be applied for the kinetic resolution of chiral spiroepoxides.And HheC-W249P is mutant of HheC ,displayed higher activity than the wide type .

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 697
  • 1000
    COMPATIBLE WITH RFC[1000]

Improvement from UESTC-China

As a mutant with high activity toward 1,3-DCP, HheC-W249P has few changes in the activity of 2,3-DCP. This year we UESTC-China used this part as the substrate to successfully obtain two mutants BBa_K2286006 (P84A) and BBa_K2286008 (P84-W249P) with improved 2,3-DCP activity by molecular simulation and single-site saturation mutagenesis.

Activity toward 2，3-DCP

Halogen ion release occurs when the haloalcohol dehalogenase catalyzes the the o-halanol to the epoxide. The interaction of the halide with Hg2+ allows Hg2+ to be separated from SCN-, which forms a colored complex with Fe3 +. This complex can be determined by absorption at 460 nm. Therefore, we determined these two mutants activity by the halide ion method.


File:Https://static.igem.org/mediawiki/2017/2/2b/T--UESTC-China--W249P-P84A-W.png
Figure 1.The activity of mutants on 2,3-DCP Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃


The results show that the catalytic activity of mutant P84A to 2,3-DCP is 2.42 times that of W249P, and the catalytic activity of P84A-W249P to 2,3-DCP is 1.15 times that of W249P at 37℃ and pH 8.5.

Activity toward other substrates

haloalcohol dehalogenase HheC can catalyze o-halide into epoxides and hydrogen halides through intramolecular nucleophilic substitution mechanisms, which are involved in the catalytic degradation of many halogen compounds and have a wide range of catalytic substrates. Therefore, in order to clarify whether the high activity of the mutants are suitable for other substrates, under the same conditions, we select 1,3- dichloropropane-2-ol (DCP) and 3-chloropropane-1,2-diol (CPD) as the substrate for further detection. The results show that the activity of P84A-W249P toward both substrates is lower than W249P. And the activity of P84A toward CPD has a certain increase. For 1,3-DCP, its activity is lower than W249P, but still has a very high activity.

Figure 2. The activity of mutants on CPD

Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃

Figure 3. The activity of mutants on 1,3-DCP

Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃

The above experimental results show that 84 amino acid of HheC has an important effect on its catalytic activity toward the 2,3-DCP, 1,3-DCP and CPD.Compared with W249P, the mutant P84A-W249P successfully improved its catalytic activity toward 2,3-DCP. While the mutant P84A not only improves its catalytic activity toward 2,3-DCP, but also maintains its high activity for 1,3-DCP and CPD.